1. Field of the Invention
The present invention relates to the microbiological industry, and specifically to a method for manufacturing a product of a reaction catalyzed by a protein having 2-oxoglutarate-dependent enzyme activity such as 4-hydroxy-L-isoleucine or a salt thereof using a bacterium transformed with a DNA fragment containing a gene coding for a protein having a 2-oxoglutarate-dependent enzyme such as L-isoleucine dioxygenase. This bacterium has also been modified to have enhanced expression of a gene coding for an L-isoleucine transporter, and has the ability to produce (2S,3R,4S)-4-hydroxy-L-isoleucine.
2. Brief Description of the Related Art
4-hydroxy-L-isoleucine is an amino acid which can be extracted and purified from fenugreek seeds (Trigonella foenum-graecum L. leguminosae). 4-hydroxy-L-isoleucine displays an insulinotropic activity, which is of great interest because of its stimulating effect which is clearly dependent on the plasma glucose concentration in the medium. This effect has been demonstrated both in isolated perfused rat pancreas and human pancreatic islets (Sauvaire, Y. et al, Diabetes, 47: 206-210, (1998)). Such a glucose dependency has not been confirmed for sulfonylurea (Drucker, D. J., Diabetes 47: 159-169, (1998)), which is the only insulinotropic drug currently used to treat type II diabetes [or non-insulin-dependent diabetes (NIDD) mellitus (NIDDM)], and as a consequence, hypoglycemia is still a common undesirable side effect of sulfonylurea treatment (Jackson, J., and Bessler, R. Drugs, 22: 211-245; 295-320, (1981); Jennings, A. et al. Diabetes Care, 12: 203-208, (1989)). Methods for improving tolerance to glucose are also known (Am. J. Physiol. Endocrinol., Vol. 287, E463-E471, 2004). This glucometabolism enhancement activity, and its potential application to pharmaceuticals and health foods, have been previously reported (Japanese Patent Application Laid-Open No. Hei 6-157302, US2007-000463A1).
4-hydroxy-L-isoleucine is only found in plants, and due to its particular insulinotropic action, might be considered a novel secretagogue with potential applications for the treatment of type II diabetes, which is characterized by defective insulin secretion associated with various degrees of insulin resistance (Broca, C. et al, Am. J. Physiol. 277 (Endocrinol. Metab. 40): E617-E623, (1999)).
Oxidizing iron, ascorbic acid, 2-oxyglutaric acid, and oxygen-dependent isoleucine by utilizing dioxygenase activity in fenugreek extract has been reported as a method for manufacturing 4-hydroxy-L-isoleucine (Phytochemistry, Vol. 44, No. 4, pp. 563-566, 1997). However, this method is unsatisfactory for manufacturing 4-hydroxy-L-isoleucine because the activity of the enzyme is inhibited by the substrate at isoleucine concentrations of 20 mM and above. Furthermore, the enzyme has not been identified, is derived from plant extracts, is not readily obtained in large quantities, and is unstable.
An efficient eight-step synthesis of optically pure (2S,3R,4S)-4-hydroxyisoleucine with 39% overall yield has been disclosed. The key steps of this synthesis involve the biotransformation of ethyl 2-methylacetoacetate to ethyl(2S,3S)-2-methyl-3-hydroxy-butanoate with Geotrichum candidum and an asymmetric Strecker synthesis (Wang, Q. et al, Eur. J. Org. Chem., 834-839 (2002)).
A short six-step chemoenzymatic synthesis of (2S,3R,4S)-4-hydroxyisoleucine with total control of stereochemistry, the last step being the enzymatic resolution by hydrolysis of a N-phenylacetyl lactone derivative using the commercially available penicillin acylase G immobilized on Eupergit C(E-PAC), has also been disclosed (Rolland-Fulcrand, V. et al, J. Org. Chem., 873-877 (2004)).
But currently, there have been no reports of producing (2S,3R,4S)-4-hydroxy-L-isoleucine by using a bacterium transformed with a DNA fragment containing a gene coding for a protein having L-isoleucine dioxygenase activity; wherein the bacterium is also modified to have enhanced expression of a gene coding for L-isoleucine transporter and has the ability to produce (2S,3R,4S)-4-hydroxy-L-isoleucine.
Besides (2S,3R,4S)-4-hydroxy-L-isoleucine, products which are produced by reactions catalyzed by proteins having 2-oxoglutarate-dependent enzyme activity, and which are industrially important are known. However, there have been no reports of systems for efficiently producing the products by using the proteins having 2-oxoglutarate-dependent enzyme activity.